First Report of Trichoderma parareesei Causing Seedling Blight on Eggplant in China.

Eggplant (Solanum melongena L.) is an economically important vegetable crop in subtropics and tropics. In March 2021, a serious disease on eggplant seedlings about 20 days after transplanting was found in Rong'an County (25°28' N; 109°53' E), Guangxi, China, with an incidence of diseased plants of 35%. The initial symptom was water-soaked spots on the leaves, followed by irregular black-brown spots that gradually expanded outward, causing leaf necrosis and defoliation. Even parts of eggplant seedlings died after lesions extended to the stem and the surface of the diseased tissues was covered with white to blue mold. Four diseased eggplants were randomly collected from different fields. Small pieces of the symptomatic tissues were surface sterilized and incubated on potato dextrose agar (PDA) at 28°C for 4 days. A total of 12 strains with similar morphological characteristics were isolated, and four representative strains (FW-01 to FW-04) were characterized. The colony was initially white, changing to yellow-green after 7 days. Phialides were lageniform or ampulliform, 2.9 to 9.75 µm × 1.36 to 4.3 µm (n=50). Conidia were green, ellipsoidal to oblong, smooth, 2.1 to 3.3 µm × 1.6 to 2.33 µm (n=50). Chlamydospores were not observed on PDA. These morphological characteristics are consistent with the description of the genus Trichoderma (Samuels et al. 2012). To confirm the identification, from mycelia of the four isolates and DNA was extracted using the Fungal Genomic DNA Extraction Kit (Bioer Technology [Hangzhou] Co., Ltd.). Three gene regions (ITS, tef1 and rpb2) were amplified (Sadfi-Zouaoui et al. 2009; Atanasova et al. 2010) and sequenced (GenBank Accessions: OL677389 to OL677392 for ITS, OL743178 to OL743181 for tef1 and OL743182 to OL743185 for rpb2). ITS sequences shared 100% identity with sequences of T. reesei (MW514156) and T. parareesei (HM466668), and tef1 and rpb2 sequences showed more than 99% similarity with sequences of T. parareesei (KM263190 and HM182962). The phylogenetic tree of the concatenated sequences showed that four isolates were clustered with T. parareesei. Therefore, the isolates were identified as T. parareesei. To satisfy Koch's postulates, the pathogenicity of four strains was tested on healthy eggplant seedlings planted in a sterile potting mix. Eggplants at four leaves stage were inoculated using conidial suspensions (with a concentration of 1 × 106 conidia/ml), with two leaves of each eggplant inoculated with each isolate and the test repeated three times. The control eggplants leaves were inoculated with sterile water. All plants were placed in a greenhouse at 22 ± 3°C and 85% relative humidity, with a photoperiod of 12 h. The water-soaked spots appeared 48 h after inoculation. All inoculated leaves showed symptoms 3 days post-inoculation. The diseased leaves became brittle and abcissed, while the control leaves remained symptomless. Only T. parareesei was successfully re-isolated from the lesions. Atanasova et al. (2010) found that T. parareesei inhibited the growth of Lepidium sativum seedlings under in vitro conditions (Atanasova et al. 2010). To our knowledge, this is the first report of T. parareesei causing eggplant seedling blight in China. The pathogen can cause substantial economic losses in eggplant production. Therefore, the identification of the pathogen is of great significance for the diagnosis and control of the disease. The results of this study deepen the understanding of the pathogenicity of Trichoderma.

First Report of Stem and Leaf Blight Disease on Mesona chinensis Caused by Rhizoctonia solani in China.

Mesona chinensis is an important medicinal and edible plant resource distributed in eight provinces in southern China. In December 2021, an unknown stem and leaf blight disease was found in M. chinensis cultivation areas in Longzhou County, Guangxi, China. Sixty days after transplanting, the incidence of this disease was 10%. Leaf spots mostly appeared from the leaf edge, were irregular, brown to dark brown, causing more than half of the leaf or the whole leaf to die. The infected stem first showed dark brown spots, then constricted slightly, became necrotic and rotted with the expansion of the spots, resulting in the death of the whole plant. Loose cobweb-like mycelia, which resembled Rhizoctonia, could be seen on the diseased tissues in conditions of high humidity. To identify the pathogen, diseased stems and leaves with typical symptoms from Longzhou County were collected and surface-sterilized with 75% ethanol for 30 s. Small fragments (5×5 mm) at the junction of diseased and healthy tissues were disinfected with 1% NaClO for 1min, washed with sterile water three times, transferred to potato dextrose agar (PDA), and incubated at 28°C for 3 days. Mycelial tips were removed, and six isolates (No. R1-R6) were obtained. The colonies were initially gray white and later light brown. Many nearly round to irregular sclerotia appeared after 7 days of culture. The sclerotia turned from light brown to deep brown and were 1 to 5 mm in diameter. The mycelium branched at a 90° angle, with septa near the branches and a constriction of the mycelium at the base of the branch. These morphological characteristics were consistent with Rhizoctonia. For molecular identification, genomic DNA of the six isolates was obtained using an extraction kit (Biocolor, Shanghai, China), and primers ITS4/ITS5 were used to amplify the internal transcribed spacers (ITS) and 5.8S rRNA (White et al. 1990). A 750 bp DNA fragment was obtained and the sequences were deposited in GenBank (OM095383-OM095388). All isolates had ≥ 99% identity with anastomosis group AG1-1B (HG934429 and HQ185364) of R. solani. A phylogenetic tree showed that the isolates and those from anastomosis group AG1-1B clustered into one branch. To satisfy Koch's postulates, the isolates from diseased leaf (No. R1, R2, and R3) and diseased stem (No. R4, R5, and R6) were inoculated on leaves and stems of 45-day-old M. chinensis plants. Five leaves and stems were inoculated with mycelial plugs of each isolate without wounding and another five leaves and stems were inoculated with mycelial plugs of each isolate after pinprick wounding. Control wounded leaves and stems were inoculated with sterile PDA discs. To maintain high humidity, the plants were incubated at 28°C and covered with transparent plastic covers. Diseased spots first appeared 24 h after inoculation. Three days post-inoculation, all inoculated leaves and stems showed symptoms like those observed in the field, whereas controls were asymptomatic. The pathogen was re-isolated from the diseased inoculated tissues using the method described above, and isolated fungi had the morphological characteristics of R. solani. Thus, the pathogen causing stem and leaf blight disease of M. chinensis was determined to be R. solani. The host range of R. solani is wide, and anastomosis group AG1-1B has been reported to infect plants such as rice, bean, fig, cabbage, and lettuce (Sneh et al. 1991). To our knowledge, this is the first report of R. solani causing a stem and leaf blight on M. chinensis, and provides a basis for diagnosis and control of the disease.

First report of leaf spot caused by Colletotrichum siamense on Michelia alba in China.

Michelia alba (common name: white champaca), native to Indonesia, is a preciously ornamental and medicinal plant in the west and southeast of China and widely distributed in Nanning, Guangxi, China (Hou et al. 2018). In May 2020, a foliar disease of M. alba was observed in Nanning (22°51' N; 108°17' E), Guangxi, China, present on ca. 20-30% of the leaves. The disease began to develop from the margins of leaves in most cases. The symptoms recorded were light yellow spots, which gradually developed into ellipsoidal to irregular brown spots, surrounded by a wide yellow halo. The spots gradually enlarged in size and became grey-brown, with the dimension of 3.5 × 2.8 to 11.0 × 3.5 cm, even more than half of leaf area. In the later stage of infection, these spots coalesced resulting in necrosis and early shedding of the leaves. Sometimes black acervuli were observed on some lesions. For isolation of the fungus, ten symptomatic leaves were randomly sampled from five trees and washed with sterile water. Small pieces of infected tissue (about 4 mm2) were surface disinfected in 75% alcohol for 30 s and in 0.1% aqueous solution of mercury chloride for 1 min. Finally these tissue pieces were rinsed three times with sterile water, plated on potato dextrose agar (PDA) and then incubated for 7 days at 28℃ with a photoperiod of 12 h. Fifteen strains with similar morphological characterizations were isolated, and five representative isolates (BL-1 to BL-5) were purified. These cultures gave rise to grey-white colonies with bright orange conidial masses with contained one-celled, hyaline, guttulate conidia, measuring 12.68-20.70 × 4.27-7.84 µm (average 15.36 × 5.35 µm, n=100). Appressoria formed from conidia were brown, ellipsoidal or inverted trapezoid and measured 6.36-12.13 × 5.07-7.39 µm (average 8.29 × 6.36 µm, n=30). These morphological characteristics were similar to those of the Colletotrichum gloeosporioides species complex (Weir et al. 2012). To confirm identification, genomic DNA from mycelium of these five isolates was extracted, and the sequence of internal transcribed spacer (ITS), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), calmodulin (CAL) and ß-tubulin (TUB2) were amplified (Zhang et al. 2020), and the GenBank accession numbers for the sequences were MW186173 to MW186177 (ITS), MW161290 to 161294 (CHS-1), MW161295 to MW161299 (GAPDH), MW161285 to 161289 (ACT), MW084710 to 084714 (CAL) and MW161300 to MW161304 (TUB2). The phylogenetic tree of six combined genes of the five isolates clustered with Colletotrichum siamense strains (CBS 125378, ICMP 17795 and ICMP 18121). Therefore, the isolates were identified as C. siamense. Five isolates (BL-1 to BL-5) were tested for pathogenicity. Wounded and unwounded detached healthy leaves were inoculated using mycelial discs (5 mm in diameter) and conidial suspensions (with the concentration of 1 × 105 conidia/ml) at the same time, incubated in a growth chamber at 25-30℃ (85-90% relative humidity, with a photoperiod of 12 h). Three leaves (wounded left half blade and unwounded right half blade) were inoculated with different methods for each isolate, and the tests were repeated three times. Four days after inoculation, leaf spots were observed on all wounded leaves, while 5-10% of the unwounded leaves showed lesions. Control leaves inoculated with PDA discs and sterile water remained symptomless. Colletotrichum. siamense was re-isolated from the lesions, confirming Koch's postulates. At least 60 plant species have been reported to be infected by C. siamense worldwide (Ji et al. 2019). To our knowledge, this is the first report of C. siamense causing leaf spot on M. alba in China.

First Report of Stagonosporopsis vannaccii Causing Leaf Spot on Crassocephalum crepidioides in China.

Crassocephalum crepidioides (Benth.) S. Moore, native to tropical Africa, is an important invasive weed in many countries, seriously threatening the safety of agricultural ecosystem. During December 2018, 100% of C. crepidioides plants exhibited leaf spots in the Kudzu (Pueraria lobata) garden in Tianlin County, Baise City, Guangxi, China (24°40'20.42″N, 106°11'33.51″E), but Kudzu was not affected by this disease. The leaf spots appeared as small brown spots surrounded by a yellow-green halo initially, enlarged to subrotund or irregular in shape, slightly sunken, then developed as a dark brown to dark spot with grey-white necrotic center (Supplementary Fig. 1 a,b), and exuded an orange droplet under high humidity conditions (Supplementary Fig. 1 c). Symptomatic leaf tissues were cut into small pieces (5 x 5 mm) from the junction of necrotic and healthy tissues, and small pieces were disinfected in 75% ethanol solution for 30 s and 0.1% mercury dichloride for 30 sec, then rinsed with sterile water 3 times. These tissues were plated onto potato dextrose agar (PDA) medium and incubated in a thermostatic incubator at 28°C under natural sunlight conditions. Four isolates with similar morphological features were obtained after purification. Colonies of these isolates exhibited creme-orange margins and aerial mycelium was sparse. The colonies formed concentric circles on the surface that were fusco-black, violet-slate and vinaceus-grey (from centre to edge), fusco-black on the reverse after 7 days (Supplementary Fig. 2 a,b), and then the pycnidia and conidia produced for about 30 days (Supplementary Fig. 2 c). Pycnidia of representative isolate YTH-12 were black, subglobose, and unilocular, 95.60-168.27 µm (average 128.32 µm) (n = 40) in diameter. The ostiole was single and central, slightly papillate to papillate and occasionally rostrate (Supplementary Fig. 2 d). Conidia were hyaline, oval to elliptical, aseptate, 2.30 to 5.83 × 1.42 to 3.50 µm (average, 4.36 × 2.03 µm) (n = 50) (Supplementary Fig. 2 e). These morphological characters are consistent with those described for Stagonosporopsis vannaccii (Crous et al. 2019). To further identify the isolate YTH-12, the rDNA internal transcribed spacer (ITS) region, 28s large subunit ribosomal RNA (LSU), RNA polymerase II second largest subunit (RPB2) gene and ß-tubulin (TUB2) gene were amplified by polymerase chain reaction using the primer pairs ITS1/ITS4 (White et al. 1990), LR0R (Rehner and Samuels 1994)/LR7 (Vilgalys and Hester 1990), Btub2Fd/Btub4Rd (Woudenberg et al. 2009) and RPB2-5F2 (Sung et al. 2007)/fRPB2-7cR (Liu et al. 1999), respectively. The PCR products were purified and sequenced by Sangon Biotech Co. Ltd. (Shanghai, China). The sequences were deposited in GenBank (accession nos. MN892355, MN893911, MN905510 and MN905511). The ITS (522 bp), LSU (1313 bp), TUB2 (380 bp) and RPB2 (1193 bp) nucleotide sequences showed 100% identity to S. vannaccii strain LFNO148 (accession nos. MK519453, MK519452, MK519454 and MN534891). Phylogenetic analysis based on the multi-locus sequences of ITS, LSU, RPB2 and TUB2 was performed in MEGA version 6.0 (Chen et al. 2015). The relative stability of the branches was evaluated by bootstrapping with 1000 replications. The isolate YTH-12 was placed in the same clade as S. vannaccii with 100% bootstrap support. Based on morphology and molecular analyses, this pathogen was identified as S. vannaccii. To satisfy Koch's postulates, the isolate YTH-12 was inoculated on leaves of C. crepidioides plants. Twenty punctured leaves and twenty unwounded leaves were inoculated with a 5-mm-diameter mycelial disc, respectively. Leaves inoculated with sterile PDA discs were used as blank controls. Plants were maintained in a growth chamber (25°C-28°C and relative humidity 80%-90%). Brown spots were observed on inoculated leaves (both punctured and unwounded) about 30 hours after inoculation and typical symptoms appeared about 55 hours after inoculation (Supplementary Fig. 1 d), and the diseased leaves produced black pycnidia and orange droplet 10 days after inoculation (Supplementary Fig. 1 e). All inoculated leaves developed symptoms similar to those on the naturally infected plants in the garden and the disease incidence reached 100%, whereas the control leaves remained symptomless (Supplementary Fig. 1 f). The same fungus was re-isolated from inoculated leaves. To our knowledge, this is the first report of S. vannaccii causing leaf spot on C. crepidioides in China. So far, Stagonosporopsis vannaccii has been reported as a plant pathogenic fungus only in Brazil, causing anthracnose symptoms on pods of soybean (Crous et al. 2019). Crassocephalum crepidioides is a widely distributed weed. If S. vannaccii has strong host specificity, it is possible to be used as a biocontrol fungus to control the weed. Conversely, if the fungus has a wider host range, C. crepidioides may act as a good bridge to spread the pathogen. This study helps to deepen the understanding of S. vannaccii and its associated plant diseases.